Plant GH3 proteins are a group of enzymes whose activity is tightly linked with hormone homeostasis and signaling pathways, and therefore their complete characterization is of major relevance for a better understanding of the influence of hormones in plant responses and development programs. According to sequence similarities and substrate specificities, the family is divided in three major subfamilies. In the present work, we have identified a new GH3 Subfamily (IV) with specific sequence characteristics that suggests activity over non-yet described substrates. Our results show that this Subfamily is absent in relevant species like Arabidopsis or maize. The gene promoter analysis of members of this Subfamily in several woody species implies that the activity of these proteins is mainly related to light responses, biotic and abiotic stresses, among other processes. Characterization of the substrates of these enzymes is necessary for a complete understanding of the activity of the GH3 family.